Automated DNA extraction using cellulose magnetic beads can improve <i>EGFR</i> point mutation detection with liquid biopsy by efficiently recovering short and long DNA fragments

Abstract

// Chiho Nakashima 1 , Akemi Sato 2 , Tomonori Abe 1 , Junichi Kato 3 , Mitsuharu Hirai 3 , Tomomi Nakamura 1 , Kazutoshi Komiya 1 , Eisaburo Sueoka 2 , Shinya Kimura 1 and Naoko Sueoka-Aragane 1 1 Division of Hematology, Respiratory Medicine and Oncology, Department of Internal Medicine, Faculty of Medicine, Saga University, Saga, Japan 2 Department of Clinical Laboratory Medicine, Faculty of Medicine, Saga University, Saga, Japan 3 ARKRAY, Inc., Kyoto, Japan Correspondence to: Naoko Sueoka-Aragane, email: sueokan@cc.saga-u.ac.jp Keywords: liquid biopsy; EGFR mutation; DNA extraction; pre-analytical procedure; plasma DNA integrity Received: November 22, 2017&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Accepted: April 28, 2018&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Published: May 18, 2018 ABSTRACT The clinical utility of plasma DNA for detecting cancer-specific mutations has rapidly achieved recognition, but reliability has not been established because of relatively low mutation-detection rates compared with those from tissue re-biopsy. To address this shortcoming we examined efficiency, in terms of mutation detection, of an automated DNA extraction system that uses cellulose magnetic beads. A fully automated, highly sensitive point-mutation-detection method, mutation-biased PCR and quenching probe (MBP-QP) system, was used for this study. Plasma DNA was extracted from 61 plasma samples collected from patients with advanced non-small cell lung cancer. Extraction was performed manually with 200 &mu;l plasma (200-M) by using a silica membrane spin column system or an automated system using 200 &mu;l (200-A) or 1000 &mu;l (1000-A) plasma. Median DNA yield quantified by real-time PCR was 4.4, 4.5, and 17.3 ng with the three methods, respectively. Sensitivity for detecting epidermal growth factor receptor ( EGFR ) L858R point mutation was 36.6%, 58.5%, and 77.5%, and specificity was 93.3%, 100%, and 96.7%, respectively. Concordance rates were 60.6%, 76.1%, and 85.7%. The size distribution of plasma DNA with automated extraction was bimodal with modes at about 170 bp and 5 Kb, and plasma DNA of both sizes included tumor-derived DNA. In this report, we demonstrate that automated DNA extraction using cellulose magnetic beads can improve mutation-detection rates with plasma DNA in association with two overall sizes of DNA fragments recovered by this DNA isolation system. Examining the biological characteristics of these fragments will be the subject of further investigation.